Alcohol product processes

ABSTRACT

The present invention relates to processes for production of an alcohol product from granular starch comprising a pre-treatment at an elevated temperature below the initial gelatinization temperature of said granular starch followed by simultaneous saccharification and fermentation, and optionally recovery of ethanol.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/797,393 filed Mar. 10, 2004 and claims, under 35 U.S.C, 119, thebenefit of U.S. provisional application no. 60/453,326 filed on Mar. 10,2003, the contents of which are fully incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to processes for production of an alcoholproduct from granular starch comprising a pre-treatment at an elevatedtemperature below the initial gelatinization temperature of the granularstarch followed by simultaneous saccharification and fermentation.

BACKGROUND OF THE INVENTION

Granular starch is found in grains, cereals or tubers of plants. Thegranular starch is in the form of microscopic granules, which areinsoluble in water at room temperature. When an aqueous starch slurry isheated, the granules swell and eventually burst, dispersing the starchmolecules into the solution. During this “gelatinization” process, thereis a dramatic increase in viscosity. Because the solids level is 30-40%in a typical industrial process, the starch has to be thinned or“liquefied” so that it can be handled. This reduction in viscosity isgenerally accomplished by enzymatic degradation in a process referred toas liquefaction. During liquefaction, the long-chained starch isdegraded into smaller branched and linear chains of glucose units(dextrins) by an alpha-amylase.

A conventional enzymatic liquefaction process may be carried out as athree-step hot slurry process. The slurry is heated to between 80-85° C.and thermostable alpha-amylase added to initiate liquefaction. Theslurry is then jet-cooked at a temperature between 105-125° C. tocomplete gelatinization of the slurry, cooled to 60-95° C. and,generally, additional alpha-amylase is added to finalize hydrolysis. Theliquefaction process is generally carried out at pH between 5 and 6.Milled and liquefied whole grains are known as mash.

During saccharification, the dextrins from the liquefaction are furtherhydrolyzed to produce low molecular sugars DP1-3 that can be metabolizedby yeast. The hydrolysis is typically accomplished using glucoamylases,alternatively or in addition to glucoamylases, alpha-glucosidases and/oracid alpha-amylases can be used. A full saccharification step typicallylast up to 72 hours, however, it is common only to do apre-saccharification of, e.g., 40-90 minutes at a temperature above 50°C., followed by a complete saccharification during fermentation in aprocess known as simultaneous saccharification and fermentation (SSF).

Fermentation, may be performed using a yeast,, e.g., from Saccharomycesspp., which added to the mash, When the alcohol product is recoveredethanol, e.g., fuel, potable, or industrial ethanol, the fermentation iscarried out, for typically 35-60 hours at a temperature of typicallyaround 32° C. When the alcohol product is beer, the fermentation iscarried out, for typically up to 8 days at a temperature of typicallyaround 14° C.

Following fermentation, the mash may be used, e.g., as a beer, ordistilled to recover ethanol. The ethanol may be used as, e.g., fuelethanol, drinking ethanol, and/or industrial ethanol.

It will be apparent from the above discussion that the starch hydrolysisin a conventional alcohol product process is very energy consuming dueto the different temperature requirements during the various steps, U.S.Pat. No. 4.316,956 provides a fermentation process for conversion ofgranular starch into ethanol. European Patent No. 0140410 provides anenzyme composition for starch hydrolysis. The object of the presentinvention is to provide improved processes for conversion of granularstarch into alcohol products.

SUMMARY OF THE INVENTION

The present invention provides methods for producing an alcohol productfrom granular starch without prior gelatinization of said starch.Accordingly in a first aspect, the invention provides a process forproduction of an alcohol product comprising the sequential steps of: (a)providing a slurry comprising water and granular starch, (b) holdingsaid slurry in the presence of an acid alpha-amylase and a glucoamylaseat a temperature of 0° C. to 20° C. below the initial gelatinizationtemperature of said granular starch for a period of 5 minutes to 12hours, (c) holding said slurry in the presence of an acid alpha-amylase,and a glucoamylase, and a yeast at a temperature of between 10° C. and35° C. for a period of 20 to 250 hours to produce ethanol and (d)optionally recovering the ethanol.

Although not limited to any one theory of operation, the presentinvention, in particular, process step (b), is believed to result inswelling of starch granules enclosed in the plant cells resulting in thedisruption of cell walls and release of the starch granules therebyrendering the starch granules more accessible to further hydration andthe action of the enzymes, As hydration progresses through step (b), theacid alpha-amylase degrades the starch granules to produce dextrins,which are degraded by the glucoamylase into glucose. This processcontinues during step (c) in which the glucose is continuously fermentedto ethanol by the yeast, thereby maintaining the concentration offermentable sugar at a relatively low concentration throughout thefermentation. Without being limited to any one theory of operation, itis believed that due to the low concentration of sugars present duringfermentation, the production of glycerol by the yeast is decreased asthere is a limited need for glycerol for osmoregulation. In this regard,the present invention may be used to produce an alcohol product whichhas a reduced glycerol and/or methanol content compared to conventionalprocesses.

The present invention provides a less energy consuming alternative toconventional processes which must employ significant amounts of energyto gelatinize the starch slurry. Other advantages of the presentinvention include, without limitation, the ability to employ a low pHthroughout the process, thus reducing the risk of unwanted microbialgrowth, and reducing or eliminating the need for expensive equipment togelatinize the starch, such as, jetting installations and steam plantequipment.

In a second aspect the present invention relates to an enzymecomposition comprising an acid alpha-amylase and a glucoamylase, whereinthe ratio between the acid alpha-amylase activity and glucoamylaseactivity is from 0.30 to 5.00 AFAU/AGU wherein an additional enzymeactivity is present said enzyme activity is selected from the listconsisting of cellulase, xylanase and phytase.

In a third aspect the present invention relates to a use of the enzymecomposition of the second aspect in an alcohol product process or astarch hydrolysis process.

In a fourth aspect the present invention relates to a use of an enzymecomposition comprising an acid alpha-amylase and a glucoamylase, whereinthe ratio between the acid alpha-amylase activity and glucoamylaseactivity is from 0.30 to 5.00 AFAU/AGU, in an alcohol product processcomprising hydrolysis of granular starch.

In a sixth aspect the present invention relates to a mashing processcomprising application of an acid alpha-amylase.

DETAILED DESCRIPTION OF THE INVENTION

The term “alcohol product” means a product comprising ethanol, e.g.,fuel ethanol, potable and industrial ethanol. However, the alcoholproduct may also be a beer, which beer may be any type of beer.Preferred beer types comprise ales, stouts, porters, lagers, bitters,malt liquors, happoushu, high-alcohol beer, low-alcohol beer,low-calorie beer or light beer.

The term “granular starch” means raw uncooked starch, i.e., starch inits natural form found in cereal, tubers or grains. Starch is formedwithin plant cells as tiny granules insoluble in water. &hen put in coldwater, the starch granules may absorb a small amount of the liquid andswell. At temperatures up to 50° C. to 75° C. the swelling may bereversible. However, with higher temperatures an irreversible swellingcalled gelatinization begins.

The term “initial gelatinization temperature” means the lowesttemperature at which gelatinization of the starch commences. Starchheated in water begins to gelatinize between 50° C. and 75° C.; theexact temperature of gelatinization depends on the specific starch andcan readily be determined by the skilled artisan. Thus, the initialgelatinization temperature may vary according to the plant species, tothe particular variety of the plant species as well as with the growthconditions. In the context of this invention the initial gelatinizationtemperature of a given starch is the temperature at which birefringenceis lost in 5% of the starch granules using the method described byGorinstein. S. and Lii. C., Starch/Stärke, Vol. 44 (12) pp. 461-466(1992).

The polypeptide “homology” means the degree of identity between twoamino acid sequences. The homology may suitably be determined bycomputer programs known in the art, such as, GAP provided in the GCGprogram package (Program Manual for the Wisconsin Package, Version 8,August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wis.,USA 53711) (Needleman, S. B. and Wunsch, C. D., (1970), Journal ofMolecular Biology, 48, 443-453. The following settings for polypeptidesequence comparison are used: GAP creation penalty of 3.0 and GAPextension penalty of 0.1.

The term “acid alpha-amylase” means an alpha-amylase (E.C. 3.2.1.1)which added in an effective amount has activity at a pH in the range of3.0 to 7.0, preferably from 3.5 to 6.0, or more preferably from 4.0-5.0.Any suitable acid alpha-amylase may be used in the present invention.

In a preferred embodiment, the acid alpha-amylase is an acid fungalalpha-amylase or an acid bacterial alpha-amylase. Preferably the acidfungal alpha-amylase is obtained from a strain of Aspergillus,preferably a strain of Aspergillus niger or a strain of a strain ofAspergillus oryzae. More preferably the acid alpha-amylase is an acidalpha-amylase having at least 70% homology, such as at least 80% or evenat least 90% homology to the acid fungal alpha-amylase having the aminoacid sequence set forth in SEQ ID NO: 1 or having at least at least atleast 70% homology, such as at least 80% or even at least 90% homologyto the acid fungal alpha-amylase having the amino acid in the sequenceshown in SWISPROT No: P10529.

Most preferably the acid alpha-amylase is an acid fungal alpha-amylasehaving the amino acid sequence set forth in SEQ ID NO, 1 or variantsthereof having one or more amino acid residues which have been deleted,substituted and/or inserted compared to the amino acid sequence of SEQID NO: 1; which variants have alpha-amylase activity.

Preferred acid alpha-amylase for use in the present invention may bederived from a strain of B. licheniformis, B. amyloliquefaciens, and B.stearothermophilus. Also preferred are acid alpha-amylases having anamino acid sequence which has at least 50% homology, preferably at least60%, 70%, 80%, 85% or at least 90%, e.g., at least 95%,, 97%, 98%, or atleast 99% homology to the sequences set forth in SEQ ID NO: 2 or SEQ IDNO: 3. Preferably the acid alpha-amylase used for the process of theinvention is one of the acid alpha-amylase variants and hybridsdescribed in WO 96/23874, WO 97/41213, and WO 99/19467, such as theBacillus stearothermophilus alpha-amylase (BSG alpha-amylase) varianthaving the following mutations delta (181-182)+N193F (also denoted I181*+G182*+N193F) compared to the wild type amino acid sequence set forth inSEQ ID NO: 2. The acid bacterial alpha-amylase may also be a hybridalpha-amylase comprising the 445 C-terminal amino acid residues of theBacillus licheniformis alpha-amylase set forth in SEQ ID NO: 3 and the37 N-terminal amino acid residues of the alpha-amylase derived fromBacillus amyloliquefaciens set forth in SEQ ID NO: 4, which may furtherhave the substitutionsG48A+T491+G107A+H156Y+A181T+N190F+1201F+A209V+Q264S using the numberingin SEQ ID NO: 3. Also preferred are alpha-amylase variants derived fromBacillus amyloliquefaciens and having at least 50% homology, such as atleast 60%, at least 70%, at least 80%, or even 90% homology to thesequence set forth in SEQ ID NO: 4. Especially preferred are variantshaving one or more of the mutations H154Y, A181T, N190F, A209V and Q264Sand/or deletion of two residues between positions 176 and 179,preferably deletion of E178 and G179.

Preferred commercial compositions comprising alpha-amylase includeMycolase from DSM (Gist Brocades) BAN™, TERMAMYL™ SC, FUNGAMYL™,LIQUOZYME™ X and SAN™ SUPER, SAN™ EXTRA L (Novozymes A/S) and ClaraseL40,000, DEX-LO™, Spezyme FRED, SPEZYME™ AA, and SPEZYME™ DELTA AA(Genencor Int.).

A glucoamylase (E.C.3.2.1.3) to be used in the processes of theinvention may be derived from a microorganism or a plant. Preferred areglucoamylases of fungal origin such as Aspergillus glucoamylases, inparticular A. niger G1 or G2 glucoamylase (Boel et al., 1984, EMBO J3(5), p. 1097-1102). Also preferred are variants thereof, such asdisclosed in WO 92/00381 and WO 00/04136, the A. awamori glucoamylase(WO 84102921), A. oryzae (Agric. Biol. Chem., 1991, 55 (4), p. 941-949),or variants or fragments thereof. Preferred glucoamylases include theglucoamylases derived from Aspergillus niger, such as a glucoamylasehaving 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or even 90% homology tothe amino acid sequence set forth in We 00/04136 and SEQ ID NO: 13. Alsopreferred are the glucoamylases derived from Aspergillus oryzae, such asa glucoamylase having 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or even 90%homology to the amino acid sequence set forth in WO 00/04136 SEQ ID NO:2.

Other preferred glucoamylases include Talaromyces glucoamylases, inparticular derived from Talaromyces emersonii (WO 99/28448), Talaromycesleycettanus (U.S. Pat. No. Re,32,153), Talaromyces duponti, Talaromycesthermophilus (U.S. Pat. No, 4,587,215), Clostridium, in particular C.thermoamylolyticum (EP135, 138), and C. thermohydrosulfuricum (WO86/01831).

Commercially available compositions composing glucoamylase include AMG200L; AMG 300 L, SAN™ SUPER, SAN EXTRA L and AMG™ E (from NovozymesA/S); OPTIDEX™ 300 (from Genencor Int.); AMIGASE™ and AMIGASE™ PLUS(from DSM); G-ZYME™ G900, G-ZYME™ and G990 ZR (from Genencor tnt.).

An additional enzyme that may be used in the processes of the inventionincludes xylanases, cellutases and phytases.

A xylanase used according to the invention may be derived from anysuitable organism, including fungal and bacterial organisms, such asAspergillus, Disporotrichum, Penicillium, Neurospora, Fusarium andTrichoderma.

Preferred commercially available preparations comprising xylanaseinclude SHEARZYME®, BIOFEED WHEAT®, CELLUCLAST®, ULTRAFLO®, VISCOZYME®(from Novozymes A/S) and SPEZYME® CP (from Genencor Int.).

The cellulase activity (E.C. 3.2.1.4) may be a cellulase of microbialorigin, such as derivable from a strain of a filamentous fungus (e.g.,Aspergillus, Trichoderma, Humicola, Fusarium).

Commercially available preparations comprising cellulase which may beused include CELLUCLAST®, CELLUZYME®, CEREFLO® and ULTRAFLO® (fromNovozymes A/S), LAMINEX™ and SPEZYME® CP (from Genencor Int.) andROHAMENT® 7069 W (from Röhm GmbH).

A phytase used according to the invention may be any enzyme capable ofeffecting the liberation of inorganic phosphate from phytic acid(myo-inositol hexakisphosphate) or from any salt thereof (phytates).Phytases can be classified according to their specificity in the initialhydrolysis step, viz. according to which phosphate-ester group ishydrolyzed first. The phytase to be used in the invention may have anyspecificity, e.g., a 3-phytase (E.G. 3.1.3.8), a 6-phytase (E.C.3.1.3.26) or a 5-phytase (no E.C. number).

Commercially available phytases preferred according to the inventioninclude BIO-FEED PHYTASE™, PHYTASE NOVO™ CT or L (Novozymes A/S), orNATUPHOS™ NG 5000 (DSM).

Another enzyme used in the process may be a debranching enzyme, such asan isoamylase (E.C. 3.2.1.68) or a pullulanase (S.C. 3.2.1.41).Isoamylase hydrolyzes alpha-1,6-D-glucosidic branch linkages inamylopectin and beta-limit dextrins and can be distinguished frompullulanases by the inability of isoamylase to attack pullulan, and bythe limited action on alpha-limit dextrins. Debranching enzyme may beadded in effective amounts well known to the person skilled in the art.

In a first preferred embodiment of the first aspect, the inventionprovides a process for production of ethanol, comprising the steps of,(a) providing a slurry comprising water and granular starch, (b) holdingsaid slurry in the presence of an acid alpha-amylase and a glucoamylaseat a temperature of 0° C. to 20° C. below the initial gelatinizationtemperature of said granular starch for a period of 5 minutes to 12hours, (c) holding said slurry in the presence of an acid alpha-amylase,and a glucoamylase, and a yeast at a temperature of between 30° C. and35° C. for a period of 20 to 200 hours to produce ethanol, and (d)recovering the ethanol. The steps (a), (b), (c) and (d) are performedsequentially; however, the process may comprise additional steps notspecified in this description which are performed prior to, between orafter any of steps (a), (b), (c) and (d),

In the first preferred embodiment of the first aspect the temperatureunder step (c) is between 28° C. and 36° C., preferably from 29° C. and35° C., more preferably from 30° C. and 34° C., such as around 32° C.and the slurry is held in contact with the acid alpha-amylase, theglucoamylase and the yeast for a period of time sufficient to allowhydrolysis of the starch and fermentation of the released sugars duringstep (c), preferably for a period of 25 to 190 hours, preferably from 30to 180 hours, more preferably from 40 to 170 hours, even more preferablyfrom 50 to 160 hours, yet more preferably from 60 to 150 hours, even yetmore preferably from 70 to 140 hours, and most preferably from 80 to 130hours, such as 85 to 110 hours,

In a second preferred embodiment of the first aspect, the inventionprovides a process for production of a beer, comprising the steps of:(a) providing a slurry comprising water and granular starch, (b) holdingsaid slurry in the presence of an acid alpha-amylase and a glucoamylaseat a temperature of 0° C to 20° C. below the initial gelatinizationtemperature of said granular starch for a period of 5 minutes to 12hours, (c) holding said slurry in the presence of an acid alpha-amylase,and a glucoamylase, and a yeast at a temperature between 10° C. and 18°C. for a period of 20 to 200 hours to produce ethanol. The steps (a),(b), and (c) are performed sequentially, however, the process maycomprise additional steps not specified in this description which areperformed prior to, between or after any of steps (a), (b), and (c),

In the second preferred embodiment of the first aspect the temperatureunder step (c) is between 10° C. and 18° C., preferably from 11° C. and17° C., more preferably from 12° C. and 16° C., such as between 13° C.and 15° C., e.g. around 14° C. and the slurry is held in contact withthe acid alpha-amylase, the glucoamylase and the yeast for a period oftime sufficient to allow hydrolysis of the starch and fermentation ofthe released sugars during step (c), preferably for a period of 100 to230 hours, preferably from 150 to 210 hours, more preferably from 170 to200 hours.

The enzyme activities may preferably be dosed in form of the compositionof the second aspect of the invention.

The acid alpha-amylase is preferably an acid bacterial alpha-amylaseand/or an acid fungal alpha-amylase and/or a variant of an acidalpha-amylase derived from a bacterial and/or a fungal source.

The acid alpha-amylase is added in an effective amount, which is aconcentration of acid alpha-amylase sufficient for its intended purposeof converting the granular starch in the starch slurry to dextrins,Preferably the acid alpha-amylase is present in an amount of 10-10000AFAU/kg of DS, in an amount of 500-2500 AFAU/kg of DS, or morepreferably in an amount of 100-1000 AFAU/kg of DS, such as approximately500 AFAU/kg DS. When measured in AAU units the acid alpha-amylaseactivity is preferably present in an amount of 5-500000 AAU/kg of DS, inan amount of 500-50000 AAU/kg of DS, or more preferably in an amount of100-10000 AAU/kg of DS, such as 500-1000 AAU/kg DS.

The glucoamylase is added in an effective amount, which is aconcentration of glucoamylase amylase sufficient for its intendedpurpose of degrading the dextrins resulting from the acid alpha-amylasetreatment of the starch slurry. Preferably the glucoamylase activity ispresent in an amount of 20-200 AGU/kg of DS, preferably 100-1000 AGU/kgof DS, or more preferably in an amount of 200-400 AGU/kg of DS, such as250 AGU/kg DS, When measured in AGI units the glucoamylase activity ispreferably present in an amount of 10-100000 AGI/kg of DS, 50-50000AGI/kg of DS, preferably 100-10000 AGI/kg of DS, or more preferably inan amount of 200-5000 AGI/kg of DS.

Preferably the activities of acid alpha-amylase and glucoamylase arepresent in a ratio of between 0.3 and 5.0 AFAU/AGU. More preferably theratio between acid alpha-amylase activity and glucoamylase activity isat least 0.35, at least 0.40, at least 0.50, at least 0.60, at least0.7, at least 0.8, at least 0.9, at least 1 0, at least 1.1, at least1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least1.7, at least 1.8, at least 1.85, or even at least 1.9 AFAU/AGU.However, the ratio between acid alpha-amylase activity and glucoamylaseactivity should preferably be less than 4,5, less than 4.0, less than3,5, less than 3.0, less than 2.5, or even less than 2.25 AFAU/AGU. InAUU/AGI the activities of acid alpha-amylase and glucoamylase arepreferably present in a ratio of between 0.4 and 6.5 AUU/AGI. Morepreferably the ratio between acid alpha-amylase activity andglucoamylase activity is at least 0.45, at least 0.50, at least 0.60, atleast 0.7, at least 0.8, at least 0.9, at least 1.0, at least 1.1, atleast 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, atleast 1.7. at least 1.8, at least 1.9, at least 2.0, at least 2.1, atleast 2.2, at least 2.3, at least 2.4, or even at least 2.5 AUU/AGI.However, the ratio between acid alpha-amylase activity and glucoamylaseactivity is preferably less than 6.0, less than 5.5, less than 4.5, lessthan 4.0, less than 3.5, or even less than 3.0 AUU/AGI,

In a preferred embodiment of the first aspect of the invention the step(b) and/or step (c) is performed in the presence of an additional enzymeactivity selected from the list consisting of xylanase, cellulase andphytase. The additional enzyme is preferably added together with theacid alpha-amylase and the glucoamylase. Xylanases may be added inamounts of 1-50000 FXU/kg DS, preferably 5-5000 FXU/kg DS, or morepreferably 10-500 FXU/kg DS. Cellulases may be added in the amounts of0.01-500000 EGU/kg DS, preferably from 0.1-10000 EGU/kg DS, preferablyfrom 1-5000 EGU/kg DS, more preferably from 10-500 EGU/kg DS and mostpreferably from 100-250 EGU/kg DS. The dosage of the phytase may be inthe range 0.5-250000 FYT/kg DS, particularly 1-100000 FYT/kg DS,preferably in the range from 5-25000 FYT/kg DS, preferably 10-10000FYT/kg, such as 100-1000 FYT/kg DS,

In a preferred embodiment the starch slurry comprises water and 5-60% DS(dry solids) granular starch, preferably 10-50% DS granular starch, morepreferably 15-40% DS, especially around 20-25% DS granular starch. Thegranular starch to be processed in the processes of the invention may inparticular be obtained from tubers, roots, stems, cobs, legumes, cerealsor whole grain. More specifically the granular starch may be obtainedfrom corns cobs, wheat, barley, rye, milo, sago, cassava, tapioca,sorghum, rice, peas, bean, banana or potatoes. Preferred are both waxyand non-waxy types of corn and barley. The granular starch to beprocessed may preferably comprising milled whole grain or it may be amore refined starch quality, preferably more than 90%, 95%, 97% or 99.5%pure starch. The raw material comprising the starch is preferably milledin order to open up the structure and allowing for further processing.Dry milling as well as wet milling may be used. When wet milling isapplied it may be preceded by a soaking, or steeping step. Both dry andwet milling is well known in the art of alcohol manufacturing and ispreferred for the processes of the invention. In the second embodimentof the first aspect of the invention wherein the alcohol product is abeer the granular starch may preferably comprise at least 10%, 20%, 30%,4:0%, 50%, 60%, 70%, 80% or even at least 90% granular starch derivedfrom malted cereals, e.g., barley malt.

The pH during step (b) and/or (c) is preferably in the range of 3.0 to7.0, more preferably from 3.5 to 6.0, or most preferably from 4.0-5.0,such as from 4.3 to 4.6.

The slurry is held in contact with the acid alpha-amylase andglucoamylase at an elevated temperature but below the initialgelatinization temperature for a period of time effective to render thestarch granules susceptible for enzymatic degradation (step b),preferably for a period of 5 minutes to 12 hours, preferably from 10minutes to 6 hours, more preferably from 15 minutes to 3 hours, evenmore preferably from 20 minutes to 1½ hour, such as from 30 minutes to1¼ hour, from 40 to 70 minutes, and even from 50 to 60 minutes. Thetemperature during step (b) should always be adjusted to be below theinitial gelatinization temperature of the particular granular starch tobe processed, and will typically be between 45° C. and 75° C. Accordingto the invention step (b) is conducted at a temperature from 0° C. to20° C., preferably to from 0° C. 15° C., more preferably from 0° C. to10° C. or even more preferably from 0° C. to 5° C. below the initialgelatinization temperature of the particular starch to be processed. Theactual temperature may be from 45° C. to 75° C., but is preferably from55° C. to 65° C. Preferably the temperature at which step (b) isconducted is at least 45° C., 46° C., 47° C., 48° C., 49° C. 50° C. 51°C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C. or preferablyat least 55° C., and preferably the temperature is no more than 74° C.,73° C., 72° C., 71° C., 70° C., 69° C., 68° C., 67° C., 66° C., 65° C.,64° C., 63° C. or preferably no more than 62° C.

After being subjected to the process of the first aspect of theinvention at least 85%, 86%, 87%, 88%, 89%, 90%t 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or preferably 99% of the dry solids of the granularstarch is converted into ethanol.

The ethanol may optionally be recovered. The ethanol recovery may beperformed by any conventional manner such as, e.g., distillation and maybe used as fuel ethanol and/or potable ethanol and/or industrialethanol.

In a particularly preferred embodiment the granular starch to beprocessed is derived from dry or wet milled cereal, such as wheat,barley, rye, and/or corn, the starch slurry has a DS of 20-40 percent,the temperature during step (b) is from 50° C. to 60° C., such as 55°C., the duration of step (b) is from 30 minutes to 75 minutes, such as60 minutes and step (c) is carried out for 60 to 90 hours. The acidalpha amylase is dosed at 300 to 700 AFAU/kg DS, such as 500 AFAU/kg DSand the glucoamylase is dosed at 100 to 500 AGU/kg DS, such as 250AGU/kg DS. The ratio of acid alpha amylase to glucoamylase is from 1.0to 3.0 AFAU/AGU or preferably from 1.5 to 2.5 AFAU/AGU, such asapproximately 2.0 AFAU/AGU. In AAU/AGI the ratio of acid alpha amylaseto glucoamylase is from 1.3 to 4.0 AAU/AGI or preferably from 2.0 to 2.3AAU/AGI, such as approximately 2.7 AAU/AGI.

In a second aspect the invention provides an enzyme composition whichmay be used in any process, including the process of the first aspect ofthe invention, said enzyme composition having a ratio between acidalpha-amylase activity and glucoamylase activity of at least 0.35, atleast 0.40, at least 0.50, at least 0.60, at least 0.70, at least 0.80,at least 0.90, at least 1.00, at least 1.20, at least 1.30, at least1.40, at least 1.50, at least 1.60, at least 1.70, at least 1.80, oreven at least 1.85 AFAU/AGU. Preferably said enzyme composition has aratio between acid alpha-amylase activity and glucoamylase activity isless than 5.00, less than 4.50, less than 4.00, less than 3.00, lessthan 2.50, or even less than 2.25 AFAU/AGU. Measured in AAU/AGI theratio of acid alpha amylase to glucoamylase in said enzyme compositionis at least 0.45, at least 0.50, at least 0.60, at least 0.70, at least0.80, at least 0.90, at least 1.00, at least 1.20, at least 1.30, atleast 1.40, at least 1.50, at least 1.60, at least 1.70, at least 1.80,at least 1.90, at least 2.00, at least 2.10, at least 2.20, at least2.30, at least 2.40 or even at least 2.50 AAU/AGI. Preferably saidenzyme composition has a ratio between acid alpha-amylase activity andglucoamylase activity is less than 6.50, less than 5.00, less than 4.50,less than 4.00, or even less than 3.50 AAU/AGI.

In a preferred embodiment the composition of the second aspect of theinvention further comprises an additional enzyme activity is present,said enzyme activity is selected from the list consisting of cellulase,xylanase and phytase.

ADDITIONAL APPLICATIONS

In a sixth aspect of the invention an acid alpha-amylase, such as anacid alpha-amylase derived from a fungus, preferably of the genusAspergillus, preferably from the species A. niger and most preferablyhaving at least 50%, at least 60%, at least 70%, at least 80% or even atleast 90% homology to the sequence shown in SEQ ID NO: 1 is used in abrewing process.

Brewing processes are well-known in the art, and generally involve thesteps of matting, mashing, and fermentation. In the traditional brewingprocess the malting serves the purpose of converting insoluble starch tosoluble starch, reducing complex proteins, generating color and flavorcompounds, generating nutrients for yeast development, and thedevelopment of enzymes. The three main steps of the malting process aresteeping, germination, and kilning.

Steeping includes mixing the barley kernels with water to raise themoisture level and activate the metabolic processes of the dormantkernel. In the next step, the wet barley is germinated by maintaining itat a suitable temperature and humidity level until adequatemodification, i.e., such as degradation of starch and activation ofenzymes, has been achieved. The final step is to dry the green malt inthe kiln.

Mashing is the process of converting starch from the milled barley maltand solid adjuncts into fermentable and unfermentable sugars to producewort of the desired composition. Traditional mashing involves mixingmilled barley malt and adjuncts with water at a set temperature andvolume to continue the biochemical changes initiated during the maltingprocess. The mashing process is conducted over a period of time atvarious temperatures in order to activate the endogenous enzymesresponsible for the degradation of proteins and carbohydrates. By farthe most important change brought about in mashing is the conversion ofstarch molecules into fermentable sugars. The principal enzymesresponsible for starch conversion in a traditional mashing process arealpha- and beta-amylases. Alpha-amylase very rapidly reduces insolubleand soluble starch by splitting starch molecules into many shorterchains that can be attacked by beta-amylase. The disaccharide producedis maltose.

To day the double-mash infusion system is the most widely used systemfor industrial production of beer, especially lager type beer. Thissystem prepares two separate mashes. It utilizes a cereal cooker forboiling adjuncts and a mash tun for well-modified, highly enzymaticallyactive malts. As the traditionally mashing processes utilize theendogenous enzymes of the barley malt the temperature is maintainedbelow 70° C. as inactivation of the enzymes would otherwise occur.

After mashing, when all the starch has been broken down, it is necessaryto separate the liquid extract (the wort) from the solids (spentgrains). Wort separation is important because the solids contain largeamounts of protein, poorly modified starch, fatty material, silicates,and polyphenols (tannins). The objectives of wort separation include thefollowing:

-   -   to produce clear wort,    -   to obtain good extract recovery, and    -   to operate within the acceptable cycle time.

Wort clarity, extraction recovery, and overall cycle times is greatlyaffected by the standard of the grist, e.g., the barley malt and thetypes of adjunct, as well as the applied mashing procedure.

Following the separation of the wort from the spent grains the wort maybe fermented with brewers yeast to produce a beer.

Further information on conventional brewing processes may be found in“Technology Brewing and Malting” by Wolfgang Kunze of the Research andTeaching Institute of Brewing, Berlin (VLB), 2nd revised Edition 1999,ISBN 3-921690-39-0.

An acid alpha-amylase, such as an acid alpha-amylase derived from afungus, preferably of the genus Aspergillus, preferably from the speciesA. niger, and most preferably having at least 50%, at least 60%, atleast 70%, at least 80% or even at least 90% homology to the sequenceshown in SEQ ID NO: 1 be applied in any brewing process as a supplementto the enzymes comprised in the malted and/or unmalted grain or in ahigher temperature mashing (HTM) process such as the one disclosed in WO2004/011591.

In the HTM process the temperature regime applied in the initial mashingphase ensures that the activity of the various endogenous enzymes of thebarley malt or of the adjunct is significantly reduced or eveneliminated. The high temperature process preferably comprises, forming amash comprising between 5% and 100% barley malt, adding prior to, duringor after forming the mash a mashing enzyme composition, attaining within15 minutes of forming the mash an initial incubation temperature of atleast 70° C., followed by incubation of the mash at a temperature of atleast 70° C. for a period of time, and separating the wort from thespent grains, Preferably the period of time of mashing is sufficient toachieve an extract recovery of at least 80%. The term “initialincubation temperature” is understood as the temperature regime during Sthe initial part of the incubation in question.

Thus at temperatures in the interval 70° C. to 78° C. only the barleymalt alpha- and beta-amylases will exhibit notable activity, and attemperatures above 78° C. the endogenous enzymes activity will benegligible. In such a mashing process the added mashing enzymes willthus constitute a very essential part of or all enzyme activity.According to one embodiment of the sixth aspect of the invention enzymeactivities needed for the mashing process to proceed are exogenouslysupplied and may be added to the mash ingredients, e.g., the water orthe grist before forming the mash, or it may be added during or afterforming the mash. The enzymes are preferably supplied all at one time atthe start of the process, however, one or more of the enzymes may besupplied at one or more times prior to, at the start, or during theprocess of the sixth aspect of the invention. In addition to an acidalpha-amylase (E.C. 3.2.1.1) the enzyme activities added may compriseone or more of the following activities; a protease (E.C. 3.4.),cellulase (E.C. 3.2.1.4) and a maltose generating enzyme. The maltosegenerating enzyme is preferably a beta-amylase (E.C. 3.2.1.2) or evenmore preferably a maltogenic alpha-amylase (E.C. 3.2.1.133).

In yet a preferred embodiment a further enzyme is added, said enzymebeing selected from the group consisting of laccase, lipase,glucoamylase, phospholipolase, phytase, phytin esterase, pullulanase,and xylanase.

In accordance with the sixth aspect of the invention a starch containingslurry, the mash, is obtained by mixing a grist comprising at least 5%,or preferably at least 10%, or more preferably at least 15%, even morepreferably at least 25%, or most preferably at least 35%, such as atleast 50%, at least 75%, at least 90% or even 100% (w/w of the grist)barley malt with water. Preferably at least 5%, preferably at least 10%,more preferably at least 20%, even more preferably at least 50%, atleast 75% or even 100% of the barley malt is well modified barley malt.In one embodiment the grist comprises other malted grain than barleymalt, so that at least 10%, at least 25%, preferably at least 35%, morepreferably at least 50%, even more preferably at least 75%, mostpreferably at least 90% (w/w) of the grist is other malted grain thanbarley malt.

Prior to forming the mash the malted and/or unmatted grain is preferablymilled and most preferably dry milled. In a preferred embodiment themalted and/or unmalted grain used is a husk free (or hull free) varietyor the husks are removed from the malted and/or unmalted grain beforeforming the mash. Removal of husks is preferably applied where themashing programs comprising temperatures above 75° C., such as attemperatures above 76° C., 77° C., 78° C., 79° C., 80° C., 81° C., 82°C., 83° C., 84° C., 85° C. or even above 86° C.

The water may preferably, before being added to the grist, be preheatedin order for the mash to attain the initial incubation temperature atthe moment of mash forming. If the temperature of the formed mash isbelow the initial incubation temperature additional heat is preferablysupplied in order to attain the initial incubation temperature.Preferably the initial incubation temperature is attained within 15minutes, or more preferably within 10 minutes, such as within 9, 8, 7,6, 5, 4, 2 minutes or even more preferably within 1 minute after themash forming, or most preferably the initial incubation temperature isattained at the mash forming.

The initial incubation temperature is preferably at least 70° C.,preferably at least 71° C., more preferably at least 72° C., even morepreferably at least 73° C., or most preferably at least 74° C., such asat least 75° C., at least 76° C., at least 77° C., at least 78° C., atleast 79° C., at least 80° C., at least 81° C., such as at least 82° C.A preferred embodiment of the mashing process of the sixth aspect of theinvention includes incubating the mash at the initial incubationtemperature of at least 70° C. and maintaining a temperature of at least70° C., preferably at least 71° C., more preferably at least 72° C.,even more preferably at least 73° C., or most preferably at least 74°C., such as at least 75° C., at least 76° C., at least 77° C., at least78° C., at least 79° C., at least 80° C., at least 81° C., at least 82°C., at least 83° C., at least 84° C., or at least 85° C., i.e., atemperature that never falls below 70° C. for the duration of theincubation period. During the incubation period the temperature ispreferably held below 100° C., such as below 99° C., 98° C., 97° C., 96°C., 95° C., 94° C., 93° C., 92° C., 91° C., or even below 90° C.

In the mashing process of the sixth aspect of the invention thetemperature may be held constant for the duration of the incubation, or,following a period of an essentially constant temperature (the initialincubation temperature) for the first part of the incubation thetemperature may be raised, either as a slow continuously increase, or asone or more stepwise increment(s) during the incubation, Alternativelythe temperature may be decreased during the incubation. In oneembodiment the initial incubation temperature is at least 70° C. andduring the incubation the temperature is increased with at least 1° C.,2° C., 3° C., 4° C. 5° C., 6° C., 75° C., 8° C., 9° C. or preferablywith at least 10° C., or more preferably with at least 12° C., such as15° C. In another embodiment the initial incubation temperature is atleast 75° C., or preferably at least 80° C. and the temperature isdecreased during the incubation with at least 5° C. or preferably withat least 1° C., 2° C., 3° C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C.or preferably with at least 10° C., or more preferably with at least 15°C. In a particular embodiment the incubation comprises maintaining themash at a temperature of at least 75° C., preferably at least 76° C.,more preferably at least 77° C., even more preferably at least 78° C.,such as at least 79° C., at least 80° C., at least 81° C., 82° C., 83°C., 84° C., 85° C., 86° C., 87° C., 88° C., 89° C. or at least 90° C.for a period of at least 1 minute, preferably for at least 5 minutes,more preferably for at least 15 minutes, even more preferably for atleast 20 minutes, such as at least 30 minutes, at least 40 minutes, atleast 50 minutes, at least 60 minutes, at least 90 minutes, or at least120 minutes. In another particular embodiment the incubation comprisesmaintaining the mash at a temperature of at least 75° C., preferably atleast 76° C., more preferably at least 77° C., even more preferably atleast 78° C., such as at least 79° C. at least 80° C., such as at least81° C., 82° C., 83° C., 84° C., 85° C., 86° C. 87° C., 88° C., 89° C. orat least 90° C. for at least 1% of the total incubation time, preferablyfor at least 5%, more preferably for at least 15%, even more preferablyfor at least 20%, or at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, such as for 100% of thetotal incubation time. The duration of the incubation is preferably atleast 15 minutes, typically between 30 minutes and 2½ hours, e.g., atleast 45 minutes, at least 1 hour, at least 1¼ hour, at least 1½ hour,at least 1¾ hour or at least 2 hours.

In the mashing process of the sixth aspect of the invention the gristmay in addition to barley malt preferably comprise adjunct such asunmalted barley, or other malted or unmalted grain, such as wheat, rye,oat, corn rice, milo, millet and/or sorghum, or raw and/or refinedstarch and/or sugar containing material derived from plants like wheat,rye, oat, corn, rice, milo, millet, sorghum, potato, sweet potato,cassava, tapioca, sago, banana, sugar beet and/or sugar cane. For theinvention adjuncts may be obtained from tubers, roots, stems, leaves,legumes, cereals and/or whole grain. Preferably the adjunct to be addedto the mash of the sixth aspect of the invention has gelatinizationtemperatures at or below the process temperature. If adjuncts such asrice or corn, or other adjuncts with similar high gelatinizationtemperature, are to be used in the process of the sixth aspect of theinvention, they may preferably be cooked separately to ensuregelatinization before being added to the mash of the sixth aspect of theinvention, or the gelatinized adjunct starch may be mashed separatelyfrom the mash adding appropriate enzymes to ensure saccharificationbefore being added to the mash. Methods for gelatinization andsaccharification of brewing adjuncts are well known in the arts. Adjunctcomprising readily fermentable carbohydrates such as sugars or syrupsmay be added to the barley matt mash before, during or after mashingprocess of the sixth aspect of the invention but is preferably addedafter the mashing process. Preferably a part of the adjunct is treatedwith a protease and/or a beta-glucanase before being added to the mashof the sixth aspect of the invention. During the mashing process, starchextracted from the grist is gradually hydrolyzed into fermentable sugarsand smaller dextrins. Preferably the mash is starch negative to iodinetesting, before extracting the wort.

Following the mashing step of the sixth aspect of the inventionobtaining the wort from the mash typically includes straining the wortfrom the spent grains, i.e. the insoluble grain and husk materialforming part of grist. Hot water may be run through the spent grains torinse out, or sparge, any remaining extract from the grist.

In the embodiment wherein the husks are removed from malted and/orunmalted grain comprised in the grist the wort separation may comprise acentrifugation step.

The wort produced by the mashing process of the sixth aspect of theinvention may be fermented to produce a beer. Fermentation of the wortmay include pitching the wort with a yeast slurry comprising freshyeast, i.e., yeast not previously used for the invention or the yeastmay be recycled yeast. The yeast applied may be any yeast suitable forbeer brewing, especially yeasts selected from Saccharomyces spp. such asS. cerevisiae and S. uvarum, including natural or artificially producedvariants of these organisms. The methods for fermentation of wort forproduction of beer are well known to the person skilled in the arts.

Preferred beer types comprise ales, strong ales, stouts, porters,lagers, bitters, export beers, malt liquors, happoushu, high-alcoholbeer, low-alcohol beer, low-calorie beer or light beer.

The enzymes to be applied in the sixth aspect of present inventionshould be selected for their ability to retain sufficient activity atelevated temperatures, such as at the process temperature of theprocesses, as well as for their ability to retain sufficient activityunder the moderately acid pH regime in the mash and should be added ineffective amounts. The enzymes may be derived from any source,preferably from a plant or an algae, and more preferably from amicroorganism, such as from a bacteria or a fungi.

Suitable proteases include microbial proteases, such as fungal andbacterial proteases. Preferred proteases are acidic proteases, i.e.,proteases characterized by the ability to hydrolyze proteins underacidic conditions below pH 7.

Contemplated acid fungal proteases include fungal proteases derived fromAspergillus, Mucor, Rhizopus, Candida, Coriolus, Endothia, Enthomophtra,Irpex, Penicillium, Sclerotium and Torulopsis. Especially contemplatedare proteases derived from Aspergillus niger (see, e.g., Koaze et al.,(1964), Agr. Biol. Chem. Japan, 28, 216), Aspergillus saitoi (see, e.g.,Yoshida, 1954, J. Agr. Chem. Soc. Japan, 28, 66), Aspergillus aculeatus(Hayashida et al., 1977. Agric. Biol. Chem., 42(5), 927-933, Aspergillusaculeatus (WO 95/02044), or Aspergillus oryzae, such as the pepAprotease; and acidic proteases from Mucor pusillus or Mucor miehei.

Contemplated are also neutral or alkaline proteases, such as a proteasederived from a strain of Bacillus. A particular protease contemplatedfor the invention is derived from Bacillus amyloliquefaciens and has thesequence obtainable at Swissprot as Accession No. P06832 (SEQ ID NO: 5).Also contemplated are the proteases having at least 90% homology toamino acid sequence obtainable at Swissprot as Accession No. P06832 (SEQID NO: 5) such as at least 92%, at least 95%, at least 96%, at least97%, at least 98%, or particularly at least 99%.

Further contemplated are the proteases having at least 90% homology toamino acid sequence disclosed as SEQ ID NO: 1 in the Danish patentapplications PA 2001 01821 and PA 2002 00005, such as at 92%, at least95%, at least 96%, at least 97%, at least 98%, or particularly at least99%.

Also contemplated are papain-like proteases such as proteases withinE.C. 3.4.22,* (cysteine protease), such as EC 3.4.22.2 (papain), EC3.4.22.6 (chymopapain), EC 3.4.22.7 (asclepain), EC 3.4.22.14(actinidain), EC 3.4.22.15 (cathepsin L), EC 3.4.22.25 (glycylendopeptidase) and EC 3.4.22.30 (caricain).

Proteases may be added in the amounts of 0.-1000 AU/kg dm, preferably1-100 AU/kg dm and most preferably 5-25 AU/kg dm.

The cellulase (E.C. 3.2.1.4) may be of microbial origin, such asderivable from a strain of a filamentous fungus (e.g., Aspergillus,Trichoderma, Humicola, Fusarium). Specific examples of cellulasesinclude the endo-glucanase (endo-glucanase I) obtainable from H.insolens and further defined by the amino acid sequence of FIG. 14 in WO91/17244 and the 43 kD H. insolens endo-glucanase described in WO91/17243.

A particular cellulase to be used in the processes of the sixth aspectof the invention may be an endo-glucanase, such as anendo-1,4-beta-glucanase. Contemplated are beta-glucanases having atleast 90% homology to amino acid sequence disclosed as SEQ ID NO: 1 inDanish patent application no. PA 2002 00130, such as at least 92%, atleast 95%, at least 96%, at least 97%, at least 98%, or particularly atleast 99%.

Commercially available cellulase preparations which may be used includeCELLUCLAST®, CELLUZYME®, CEREFLO® and ULTRAFLO® (available fromNovozymes A/S), LAMINEX™ and SPEZYME® CP (available from Genencor Int.)and ROHAMENT® 7069 W (available from Röhm, Germany).

Beta-glucanases may be added in the amounts of 1.0-10000 BGU/kg dm,preferably from 10-5000 BGU/kg dm, preferably from 50-1000 BGU/kg dm andmost preferably from 100-500 BGU/kg dm.

A particular alpha-amylase (EC 3.2.1.1) to be used in the processes ofthe sixth aspect of the invention may be any fungal alpha-amylase,preferably an acid alpha-amylase. Preferably the acid alpha-amylase isderived from a fungus of the genus Aspergillus, preferably from thespecies A. niger, and most preferably having at least 50%, at least 60%,at least 70%, at least 80% or even at least 90% homology to the sequenceshown in SEQ ID NO: 1 is used in a brewing process. Fungalalpha-amylases may be added in an amount of 1-1000 AFAU/kg DM,preferably from 2-500 AFAU U/kg DM, preferably 20-100 AFAU U/kg DM.

Another acid alpha-amylase enzyme to be used in the processes of thesixth aspect of the invention may be a Bacillus alpha-amylase.Well-known Bacillus alpha-amylases include alpha-amylase derived from astrain of B. licheniformis, B. amyloliquefaciens, and B.stearothermophilus. Other Bacillus alpha-amylases include alpha-amylasederived from a strain of the Bacillus sp. NCIB 12289, NCIB 12512, NCIB12513 or DSM 9375, all of which are described in detail in WO 95/26397,and the alpha-amylase described by Tsukamoto et al., Biochemical andBiophysical Research Communications, 151 (1988), pp. 25-31, In thecontext of the present invention a contemplated Bacillus alpha-amylaseis an alpha-amylase as defined in WO 99/19467 on page 3, line 18 to page6, line 27. A preferred alpha-amylase has an amino acid sequence havingat least 90% homology to SEQ ID NO. 4 in WO 99/19467, such as at least92%, at least 95%, at least 96%, at least 97%, at least 98%, orparticularly at least 99%. Most preferred variants of the maltogenicalpha-amylase comprise the variants disclosed in WO 99/43794.Contemplated variants and hybrids are described in WO 96/23874, WO97/41213, and WO 99/19467. Specifically contemplated is a recombinant B.stearothermophilus alpha-amylase variant with the mutations; I181*+G182*+N193F. Bacillus alpha-amylases may be added in the amounts of 1.0-1000NU/kg dm, preferably from 2.0-500 NU/kg dm, preferably 10-200 NU/kg dm.

Maltogenic Alpha-amylase

A particular enzyme to be used in the processes of the sixth aspect ofthe invention is a maltogenic alpha-amylase (E.C. 3.2.1.133). Maltogenicalpha-amylases (glucan 1,4-alpha-maltohydrolase) are able to hydrolyzeamylose and amylopectin to maltose in the alpha-configuration.Furthermore, a maltogenic alpha-amylase is able to hydrolyze maltotrioseas well as cyclodextrin. Specifically contemplated maltogenicalpha-amylases may be derived from Bacillus sp., preferably fromBacillus stearothermophilus, most preferably from Bacillusstearothermophilus C599 such as the one described in EP 120.693. Thisparticular maltogenic alpha-amylase has the amino acid sequence shown asamino acids 1-686 of SEQ ID NO: 1 in U.S. Pat. No. 6,162,628. Apreferred maltogenic alpha-amylase has an amino acid sequence having atleast 90% homology to amino acids 1-686 of SEQ ID NO: 1 in U.S. Pat. No.6,162,628 preferably at least 92%, at least 95%, at least 96%, at least97%, at least 98%, or particularly at least 99%. Most preferred variantsof the maltogenic alpha-amylase comprise the variants disclosed in WO99/43794.

Maltogenic alpha-amylases may be added in amounts of 0.1-1000 MANU/kgdm, preferably from 1-100 MANU/kg dm, preferably 5-25 MANU/kg dm.

Another particular enzyme to be used in the processes of the sixthaspect of the invention may be a beta-amylase (E.C 3.2.1.2).

Beta-amylases have been isolated from various plants and microorganisms(W. M. Fogarty and C. T. Kelly, Progress in Industrial Microbiology,vol. 15, pp. 112-115, 1979). These beta-amylases are characterized byhaving optimum temperatures in the range from 40° C. to 65° C. andoptimum pH in the range from 4.5 to 7.0. Specifically contemplatedbeta-amylase include the beta-amylases SPEZYME® BBA 1500, SPEZYME® DBAand OPTIMALT™ ME, OPTIMALT™, BBA from Genencor Int. as well as thebeta-amylases NOVOZYM™ WBA from Novozymes A/S. Beta-amylases may beadded in effective amounts well known to the person skilled in the art.

A further particular enzyme to be used in the processes of the sixthaspect of the invention may be a glucoamylase (E.C. 3.2.1.3) derivedfrom a microorganism or a plant. Preferred are glucoamylases of fungalor bacterial origin selected from the group consisting of Aspergillusglucoamylases, in particular A. niger G1 or G2 glucoamylase (Boel et al.(1984), EMBO J. 3 (5), p. 1097-1102), or variants thereof, such asdisclosed in WO 92/00381 and WO 00/04136; the A. awamori glucoamylase(WO 84/02921), A. oryzae (Agric. Biol. Chem., 1991, 55 (4), p. 941-949),or variants or fragments thereof. Glucoamylases may be added ineffective amounts welt known to the person skilled in the art.

Another enzyme of the process of the sixth aspect of the presentinvention may be a debranching enzyme, such as an isoamylase (E.C.3.2.1.68) or a pullulanase (E.C. 3.2.1.41). Isoamylase hydrolyzesalpha-1,6-D-glucosidic branch linkages in amylopectin and beta-limitdextrins and can be distinguished from pullulanases by the inability ofisoamylase to attack pullulan, and by the limited action on alpha-limitdextrins. Debranching enzyme may be added in effective amounts wellknown to the person skilled in the art.

MATERIALS AND METHODS

Alpha-amylase activity (KNU)

The amylolytic activity may be determined using potato starch assubstrate. This method is based on the break-down of modified potatostarch by the enzyme, and the reaction is followed by mixing samples ofthe starch/enzyme solution with an iodine solution. Initially, ablackish-blue color is formed, but during the break-down of the starchthe blue color gets weaker and gradually turns into a reddish-brown,which is compared to a colored grass standard.

One Kilo Novo alpha amylase Unit (KNU) is defined as the amount ofenzyme which, under standard conditions (i e., at 37° C.+/−0.05; 0.0003M Ca²⁺; and pH 5.6) dextrinizes 5260 mg starch dry substance MerckAmylum solubile.

A folder EB-SM-0009.02/01 describing this analytical method in moredetail is available upon request to Novozymes A/S, Denmark, which folderis hereby included by reference,

Acid Alpha-amylase Activity

When used according to the present invention the activity of any acidalpha-amylase may be measured in AFAU (Acid Fungal Alpha-amylase Units).Alternatively activity of acid alpha-amylase may be measured in AAU(Acid Alpha-amylase Units).

Acid Alpha-amylase Units (AAU)

The acid alpha-amylase activity can be measured in AAU (AcidAlpha-amylase Units), which is an absolute method. One Acid Amylase Unit(AAU) is the quantity of enzyme converting 1 g of starch (100% of drymatter) per hour under standardized conditions into a product having atransmission at 620 nm after reaction with an iodine solution of knownstrength equal to the one of a color reference.

Standard conditions/reaction conditions: Substrate: Soluble starch.Concentration approx. 20 g DS/L. Buffer: Citrate, approx. 0.13 M, pH =4.2 Iodine solution: 40.176 g potassium iodide + 0.088 g iodine/L Citywater 15°-20° dH (German degree hardness) pH: 4.2 Incubationtemperature: 30° C. Reaction time: 11 minutes Wavelength: 620 nm Enzymeconcentration: 0.13-0.19 AAU/mL Enzyme working range: 0.13-0.19 AAU/mL

The starch should be Lintner starch, which is a thin-boiling starch usedin the laboratory as calorimetric indicator. Lintner starch is obtainedby dilute hydrochloric acid treatment of native starch so that itretains the ability to color blue with iodine. Further details can befound in EP 0140410, which disclosure is hereby included by reference.

Acid Alpha-amylase Activity (AFAU)

Acid alpha-amylase activity may be measured in AFAU (Acid FungalAlpha-amylase Units), which are determined relative to an enzymestandard. 1 FAU is defined as the amount of enzyme which degrades 5.260mg starch dry matter per hour under the below mentioned standardconditions.

Acid alpha-amylase an endo-alpha-amylase(1,4-alpha-D-glucan-glucanohydrolase, E.C. 3.2.1.1) hydrolyzesalpha-14-glucosidic bonds in the inner regions of the starch molecule toform dextrins and oligosaccharides with different chain lengths. Theintensity of color formed with iodine is directly proportional to theconcentration of starch. Amylase activity is determined using reversecolorimetry as a reduction in the concentration of starch under thespecified analytical conditions.

${STARCH} + {{IODINE}\overset{{ALPHA}\text{-}{AMYLASE}}{\underset{{40{^\circ}},{{pH}\mspace{11mu} 2.5}}{}}{DEXTRINS}} + {OLIGOSACCHARIDES}$λ = 590  nm $\begin{matrix}{{blue}\text{/}{violet}} & {t = {23\mspace{14mu} {\sec.}}} & {decoloration}\end{matrix}$

Standard conditions/reaction conditions: Substrate: Soluble starch,approx. 0.17 g/L Buffer: Citrate, approx. 0.03 M Iodine (I2): 0.03 g/LCaCl₂: 1.85 mM pH: 2.50 ± 0.05 Incubation temperature: 40° C. Reactiontime: 23 seconds Wavelength: 590 nm Enzyme concentration: 0.025 AFAU/mLEnzyme working range: 0.01-0.04 AFAU/mL

A folder EB-SM-0259.02/01 describing this analytical method in moredetail is available upon request to Novozymes A/S Denmark, which folderis hereby included by reference.

Glucoamylase Activity

Glucoamylase activity may be measured in AGI units or inAmyloGlucosidase Units (AGU).

Glucoamylase Activity (AGI)

Glucoamylase (equivalent to amyloglucosidase) converts starch intoglucose. The amount of glucose is determined here by the glucose oxidasemethod for the activity determination. The method described in thesection 76-11 Starch-Glucoamylase Method with Subsequent Measurement ofGlucose with Glucose Oxidase in “Approved methods of the AmericanAssociation of Cereal Chemists”. Vol.1-2 AACC, from American Associationof Cereal Chemists (2000); ISBN: 1-891127-12-8.

One glucoamylase unit (AGI) is the quantity of enzyme which will form 1micromol of glucose per minute under the standard conditions of themethod.

Standard conditions/reaction conditions: Substrate: Soluble starch,concentration approx. 16 g dry matter/L. Buffer: Acetate, approx. 0.04M, pH = 4.3 pH: 4.3 Incubation temperature: 60° C. Reaction time: 15minutes Termination of the reaction: NaOH to a concentration ofapproximately 0.2 g/L (pH ~9) Enzyme concentration: 0.15-0.55 AAU/mL

The starch should be Lintner starch, which is a thin-boiling starch usedin the laboratory as colorimetric indicator. Lintner starch is obtainedby dilute hydrochloric acid treatment of native starch so that itretains the ability to color blue with iodine.

Glucoamylase Activity (AGU)

The Novo Glucoamylase Unit (AGU) is defined as the amount of enzyme,which hydrolyzes 1 micromole maltose per minute under the standardconditions 37° C., pH 4.3. substrate: maltose 23.2 mM, buffer: acetate0.1 M, reaction time 5 minutes.

An autoanalyzer system may be used. Mutarotase is added to the glucosedehydrogenase reagent so that any alpha-D-glucose present is turned intobeta-D-glucose. Glucose dehydrogenase reacts specifically withbeta-D-glucose in the reaction mentioned above, forming NADH which isdetermined using a photometer at 340 nm as a measure of the originalglucose concentration.

AMG incubation: Substrate: maltose 23.2 mM Buffer: acetate 0.1 M pH:4.30 ± 0.05 Incubation temperature: 37° C. ± 1 Reaction time: 5 minutesEnzyme working range: 0.5-4.0 AGU/mL

Color reaction: GlucDH: 430 U/L Mutarotase: 9 U/L NAD: 0.21 mM Buffer:phosphate 0.12 M; 0.15 M NaCl pH: 7.60 ± 0.05 Incubation temperature:37° C. ± 1 Reaction time: 5 minutes Wavelength: 340 nm

A folder (EB-SM-0131.02/01) describing this analytical method in moredetail is available on request from Novozymes A/S, Denmark, which folderis hereby included by reference.

Xylanolytic Activity

The xylanolytic activity can be expressed in FXU-units determined at pH6.0 with ramazol-xylan (4-O-methyl-D-glucurono-D-xylan dyed with RemazolBrilliant Blue R Fluka) as substrate.

A xylanase sample is incubated with the remazol-xylan substrate. Thebackground of non-degraded dyed substrate is precipitated by ethanol.The remaining blue color in the supernatant (as determinedspectrophotometrically at 585 nm) is proportional to the xylanaseactivity, and the xylanase units are then determined relatively to anenzyme standard at standard reaction conditions i.e., at 50.0° C., pH6.0, and 30 minutes reaction time.

A folder EB-SM-352.02/01 describing this analytical method in moredetail is available upon request to Novozymes A/S, Denmark, which folderis hereby included by reference.

Cellulolytic Activity

The cellulolytic activity may be measured in endo-glucanase units (EGU),determined at pH 6.0 with carboxymethyl cellulose (CMC) as substrate. Asubstrate solution is prepared, containing 34.0 g/l CMC (Hercules 7 LFD)in 0.1 M phosphate buffer at pH 6.0. The enzyme sample to be analyzed isdissolved in the same buffer. 5 ml substrate solution and 0.15 ml enzymesolution are mixed and transferred to a vibration viscosimeter (e.g.,MIVI 3000 from Sofraser, France), thermostated at 40° C. for 30 minutes.One EGU is defined as the amount of enzyme that reduces the viscosity toone half under these conditions. The amount of enzyme sample should beadjusted to provide 0.01-0.02 EGU/ml in the reaction mixture. The archstandard is defined as 830 EGU/g.

A folder EB-SM-0275.02/01 describing this analytical method in moredetail is available upon request to Novozymes A/S, Denmark, which folderis hereby included by reference.

Phytase Activity

The phytase activity is measured in FYT units, one FYT being the amountof enzyme that liberates 1 micromole inorganic ortho-phosphate per min.under the following conditions: pH 5.5, temperature 37° C.; substrate:sodium phylate (C₆H₆O₂₄P₆Na₁₂) at a concentration of 0.0050 mole/I.

Proteolytic Activity (AU)

The proteolytic activity may be determined with denatured hemoglobin assubstrate. In the Anson-Hemoglobin method for the determination ofproteolytic activity denatured hemoglobin is digested, and theundigested hemoglobin is precipitated with trichloroacetic acid (TCA).The amount of TCA soluble product is determined with phenol reagent,which gives a blue color with tyrosine and tryptophan.

One Anson Unit (AU) is defined as the amount of enzyme which understandard conditions (i.e., 25° C., pH 7.5 and 10 min. reaction time)digests hemoglobin at an initial rate such that there is liberated perminute an amount of TCA soluble product which gives the same color withphenol reagent as one milliequivalent of tyrosine.

A folder AF 4/5 describing the analytical method in more detail isavailable upon request to Novozymes A/S, Denmark, which folder is herebyincluded by reference.

Enzyme Preparations

The following enzyme preparations were used:

Bacterial alpha-amylase: An enzyme preparation comprising a polypeptidewith alpha-amylase activity (E.C. 3.2.1.1) derived from B.stearothermophilus and having the amino acid sequence disclosed as SEQID NO: 4 in WO 99/19467. Activity: 120 KNU/g (density=1.20-1.25 g/mL).

A preferred acid fungal alpha-amylase; an enzyme preparations derivedfrom Aspergillus niger comprising acid fungal alpha-amylase and someglucoamylase. Activities: 114 AFAU/g, 25 AGU/g (density=1.2 g/mL).

Glucoamylase: an enzyme preparation derived from Aspergillus nigercomprising glucoamylase and some acid fungal alpha-amylase. Activity:363 AGU/g, 86 AFAU/g (density=1.2 g/mL).

An enzyme preparation comprising xylanase and cellulase activitiesderived from Trichoderma and Aspergillus. Activity: 140 FXU/g+350 EGU/g(density=1.2 g/mL).

EXAMPLE 1

A conventional ethanol process using a traditional pre-liquefactioncalled the non-pressure cooking (NPC) is compared with the process ofthe invention, Traditional non-pressure batch cooking processes forproduction of potable alcohol is described in the Novozymes publicationNo. 2001-10782-01 entitled “Use of Novozymes enzymes in alcoholproduction”.

A 20% D.S. slurry of the milled barley grain was made in roomtemperature (RT) tap water.

The NPC pre-treatment of the conventional ethanol process was performedin 6×1-liter tubs with stirring. Bacterial alpha-amylase was added andthe tubs were placed in water bath at 65° C. When the temperature in themash reached 55° C. the heating was increased to heat the mash to 90° C.over 60 minutes. The temperature was then adjusted to 32° C. and 3×250 gmash was portioned in 500 mL blue cap flasks with air locks. To allflasks 0.25 g dry bakers yeast was added (corresponding to 5-10 millionvital cells/g mash). Enzyme activities were added according to the tablebelow and each flask was weighed. The flasks were placed in a shakingwater bath at 32° C. for 72 hours. At 48 and 72 hours the flasks wereweighed and CO₂ weight loss measured for monitoring of the fermentationprogress. The relationship used between amount of CO₂ loss and theweight of ethanol was: CO₂ loss (g)×1.045 =EtOH (g).

For the process of the invention 500 mL blue cap fermentation flaskseach with 250 g slurry was fitted with air locks. Using 6.0 N HCl the pHwas adjusted to 4.5 and glucoamylase and acid alpha-amylase was dosedaccording to table 1. The flasks were held at 55° C. for 60 minutes. Thetemperature was then adjusted to 32° C. and fermentation was performedand monitored as described above.

TABLE 1 Barley, the weight loss (g) at 48 and 72 hours. Bacterialalpha-amylase acid (KNU), acid alpha-amylase (AFAU) and glucoamylase(AGU) activity was added according to the table. Traditional non-Process of pressure cooking Low AFAU/AGU the invention KNU/kg DS 36 0 0AFAU/kg DS 39 39 540 AGU/kg DS 163 163 273 AFAU/AGU 0.24 0.24 1.98Weight loss (g), 9.0 11.0 14.7 48 hours Weight loss (g), 12.2 13.0 16.472 hours Ethanol %, 48 hours 3.76 4.60 4.60 Ethanol %, 72 hours 5.105.43 6.86 * based on weight loss at 48 and 72 hours, CO₂ loss (g) ×1.045 = EtOH (g).

EXAMPLE 2

This example illustrates the use of an enzyme composition of theinvention consisting of acid alpha-amylase, glucoamylase, cellulase andxylanase activity.

A 20% D.S. slurry of the milled barley grain was made in RT tap water.For each treatment 2×250 g was portioned in 500 mL blue cap flasks.Using 6 N HCl the pH was adjusted to 4.5. Enzymes activities were addedaccording to table 2 and 3, and a pre-treatment corresponding to step(b) of the invention was carried out for one hour at 55° C. in a shakingwater bath. The temperature was adjusted to 32° C. and 0.25 g dry bakersyeast Fermentation was performed and monitored as described above.

TABLE 2 Barley, the weight loss (g) at 48 and 72 hours. Acidalpha-amylase (AFAU), glucoamylase (AGU), cellulase (EGU) and xylanase(FXU) activity was added according to the table. Low AFAU/AGU Process ofthe invention AFAU/AGU 0.24 0.24 1.98 1.98 AFAU/kg DS 39 39 540 540AGU/kg DS 163 163 273 273 FXU/kg DS 0 70 0 70 EGU/kg DS 0 175 0 175Weight loss (g), 48 hours 9.9 11.2 12.8 14.3 Weight loss (g), 72 hours12.1 13.8 15.5 16.5 Ethanol %, 48 hours 4.14 4.68 5.35 5.98 Ethanol %,72 hours 5.06 5.77 6.48 6.90 * based on weight loss at 48 and 72 hours,CO₂ loss (g) × 1.045 = EtOH (g).

EXAMPLE 3

This example illustrates the process of the invention using various rawmaterials. A 20% D.S. slurry of the milled grain or corn meal was madein RT tab water. For each treatment 2×250 g was portioned in 500 mL bluecap flasks. Using 6 N HCl the pH was adjusted to 4.5 Enzymes were dosedaccording to table 3, 4 and 5, and a pre-treatment was carried out forone hour at 55° C. in a shaking water bath. The flasks were cooled to32° C. and 0.25 g dry bakers yeast added. The flasks were placed in awater bath at 32° C. for 72 hours (90 hours for wheat).

TABLE 3 Rye, the weight loss (g) at 48 hours and at 72 hours. Acidalpha-amylase (AFAU), and glucoamylase (AGU) activity was addedaccording to the table. AFAU/kg DS 39 540 AGU/kg DS 163 273 AFAU/AGU0.24 1.98 Weight loss (g), 48 hours 14.7 17.4 Weight loss (g), 72 hours16.2 19.2 Ethanol %, 48 hours 6.14 7.27 Ethanol %, 72 hours 6.77 8.03 *based on weight loss at 48 and 72 hours, CO₂ loss (g) × 1.045 = EtOH(g).

TABLE 4 Yellow corn meal, the weight loss (g) at 48 hours and at 72hours. Acid alpha- amylase (AFAU), and glucoamylase (AGU) activity wasadded according to the table. AFAU/kg DS 39 540 AGU/kg DS 163 273AFAU/AGU 0.24 1.98 Weight loss (g), 48 hours 12.4 14.5 Weight loss (g),72 hours 15.6 17.5 Ethanol %, 48 hours* 5.18 6.06 Ethanol %, 72 hours*6.52 7.32 *based on weight loss at 48 and 72 hours, CO₂ loss (g) × 1.045= EtOH (g).

TABLE 5 Wheat, the weight losses (g) at 48 hours and at 90 hours. Acidalpha-amylase (AFAU), and glucoamylase (AGU) activity was addedaccording to the table. AFAU/kg DS 39 540 AGU/kg DS 163 273 AFAU/AGU0.24 1.98 Weight loss (g), 48 hours 13.8 16.3 Weight loss (g), 90 hours16.7 18.5 Ethanol %, 48 hours 5.77 6.81 Ethanol %, 72 hours 6.98 7.73 *based on weight loss at 48 and 72 hours, CO₂ loss (g) × 1.045 = EtOH(g).

EXAMPLE 4

This example illustrates a process of the invention using wheat. A 20%D.S. slurry of milled wheat was made in RT tab water. For each treatment2×250 g slurry was portioned in 500 mL blue cap flasks. The pH wasadjusted to 4.5 using 6 N HCl. Enzyme activities were dosed according totable 6, and the flasks were incubated for one hour at 55.° C. in ashaking water bath. The flasks were cooled to 32° C. and 0.25 g drybakers yeast added. The flasks were placed in a water bath at 32° C. for72 hours. Weight loss data was recorded. At 50 and 72.5 hours the flaskswere weighed and CO₂ weight loss measured for monitoring of thefermentation progress. The relationship used between amount of CO₂ lossand the weight of ethanol was; CO₂ loss (g)×1.045=EtOH (g).

At 100 hours HPLC samples were drawn and the content of ethanol,methanol and glycerol was recorded.

TABLE 6 Wheat; the weight loss (g) at 50 hours and at 72.5 hours.Glucoamylase (AGU), Acid alpha-amylase (AFAU) and bacterialalpha-amylase (KNU) was added according to the table. AGU/kg DS 0.300.30 0.30 0.30 0.30 0.30 0.30 AFAU/kg DS 0.08 0.13 0.26 0.51 1.01 — —KNU/kg DS — — — — — 0.05 0.15 Weight loss (g), 50 hours 10.67 11.0111.71 12.53 14.14 14.68 15.55 Weight loss (g), 72.5 hours 12.53 12.9013.78 14.80 16.90 16.62 17.52 Ethanol % w/w, 50 hours* 4.46 4.60 4.895.24 5.91 6.14 6.50 Ethanol % w/w, 72.5 hours* 5.24 5.39 5.76 6.19 7.066.95 7.32 Ethanol % w/w, 100 hours** 6.36 6.64 7.01 7.47 7.73 8.13 8.59Methanol % w/w, 100 hours** 0.17 0.09 0.11 0.12 0.12 0.17 0.22 Glycerol% w/w, 100 hours** 0.52 0.54 0.49 0.54 0.53 0.65 0.723-methyl-1-butanol, 100 hours** 0.01 0.01 0.01 0.04 0.01 0.01 0.01*based on weight loss at 50 and 72.5 hrs, CO₂ loss (g) × 1.045 = EtOH(g) **based on HPLC at 100 hrs.

EXAMPLE 5

This example demonstrates the use of an acid alpha-amylase in a brewingprocess. The enzymes used comprised an acid fungal alpha-amylase derivedfrom Aspergillus niger having the sequence shown in SEQ ID NO: 1 analpha-amylase (E.C. 3.2.1.1) from B. stearothermophilus having the aminoacid sequence disclosed as SEQ ID NO: 4 in WO99/19467 with themutations: I181*+G182*+N193F, a glucoamylase derived from Aspergillusniger, a protease having the amino acid sequence shown as amino acidsno. 1-177 of SEQ ID NO. 2 in Danish patent applications WO 2003/048353,a cellulase (E.C. 3.2.1.4), a beta-glucanase having the amino acidsequence shown as SEQ ID NO: 1 in WO 2003/062409.

A xylanase from Aspergillus aculeatus having the sequence amino aciddisclosed as SEQ ID NO: 2 in WO 94/21785.

The acid alpha-amylase from Aspergillus niger SP288 was tested in amashing set up using both Congress mashing and Higher TemperatureMashing (HTM) conditions. The effect was evaluated on formation offermentable sugars in the wort, which is a key wort quality parameter. Abacterial heat stable alpha-amylase from Bacillus stearothermophilus wasapplied for comparison. All worts were added a xylanase 5 mg EP/kg DS,betaglucanase 5 mg EP/kg DS and protease 2.5 mg EP/kg DS.

Unless otherwise stated mashing was preformed according to ERG: 4.5.1using malt grounded according to EBC: 1.1. Mashing trials were performedin 500 ml lidded vessels each containing a mash with 50 g grist andadjusted to a total weight of 450±0.2 g with water preheated to theinitial incubation temperature +1° C. During mashing the vessels wereincubated in water bath with stirring.

One treatment comprised using Congress mashing described in EBC: 4.5.1.Thus 50.0 g malt is mixed with 200 ml water from beginning, 100 ml waterat 70° C. is added when profile reached 70° C. All mashcups arestandardized to 450.0 g at the end of mashing, which gives approximately8.6° P.

The second treatment comprised using HTM as disclosed in WO 2004/011591with the following temperature profile: an initial incubationtemperature of 70° C. for 65 minutes increasing to 90° C., with 1.0°C/min for 20 minutes, followed by 90° C. for 15 minutes and finalized bycooling with 4.5° C./min to 20° C. The recipe applied was. 50.0 g maltadded 200 ml water from beginning, at the end of mashing the mash-cupsare standardized to 300.0 g, which gives app. 13° P.

Fermentable sugars were analyzed at 8.6° P. using HPLC method equivalentto EBC: 8.7.

TABLE 7 Extract E2, extract in dry malt, % (m/m) from Congress mashing(8.6° P) and HTM (13° P). To all treatments were added xylanase 5 mgEP/kg DS, betaglucanase 5 mg EP/kg DS and Protease 2.5 mg EP/kg DS. Noadditional 75 AFAU/kg DS + 75 KNU/kg enzymes 61 AGU/kg DS DS Congressmashing 80.90 81.79 81.97 HTM 81.42 81.23 81.33 N.B. comparison onlyintended between enzyme treatments, not between methods.

TABLE 8 Overview of fermentable sugar profile from Congress mashing,8.6° P. To all treatments were added xylanase 5 mg EP/kg DS,beta-glucanase 5 mg EP/kg DS and Protease 2.5 mg EP/kg DS No additional75 AFAU/kg DS + 75 KNU/kg DS enzymes 61 AGU/kg DS Glucose 7.65 10.407.82 Maltose 43.18 43.08 43.54 Maltotriose 8.25 7.08 9.08 Fructose 3.243.29 3.40 Sum of 62.32 63.83 63.83 fermentable sugars

TABLE 9 Fermentable sugar profile from HTM mashing, 13° P. To alltreatments were added xylanase 5 mg EP/kg DS, beta-glucanase 5 mg EP/kgDS and Protease 2.5 mg EP/kg DS. No additional 75 AFAU/kg DS + enzymes61 AGU/kg DS 75 KNU/kg DS Glucose 10.19 14.12 10.43 Maltose 65.98 67.1066.46 Maltotriose 12.61 11.07 12.89 Fructose 5.27 5.15 5.13 Sum of 94.0597.44 94.91 fermentable sugars

Congress trial (a): SP288 combined with AMG showed good effect onformation of fermentable sugars, as the glucose concentration wasincreased in wort by the addition of acid alpha-amylase both compared toTermamyl SC and with out adding amylase.

Acid alpha-amylase showed very good effect on formation of fermentablesugars, predominantly the glucose concentration was increased in wort bythe addition of acid alpha-amylase both compared to Termamyl SC and without adding amylase. Overall the sum of fermentable sugars is increasedfrom 94.91 g/L to 97.44 g/L from performance of acid alpha-amylase. Thismeans that an increased part of the extract is now fermentable, whichwill yield higher alcohol amounts.

1-37. (canceled)
 38. A process for production of an alcohol productcomprising the sequential steps of: (a) holding a slurry comprisingwater and granular starch in the presence of an acid alpha-amylase and aglucoamylase at a temperature of 0° C. to 20° C. below the initialgelatinization temperature of the granular starch for a period between 5minutes and 12 hours, and (b) fermenting the slurry in the presence ofan acid alpha-amylase, a glucoamylase and a yeast at a temperaturebetween 10° C. and 35° C. to produce the alcohol product, wherein step(a) and/or step (b) is performed in the presence of a phytase.
 39. Theprocess of claim 38, further comprising recovering the alcohol product.40. The process of claim 38, wherein the alcohol product is fuelethanol, potable ethanol and/or industrial ethanol.
 41. The process ofclaim 38, wherein the temperature during step (b) is between 28° C. and36° C.
 42. The process of claim 38, wherein the pH during step (a) is inthe range of 3-7.
 43. The process of claim 38, wherein the pH duringstep (b) is in the range of 3-7.
 44. The process of claim 38, whereinthe starch slurry has 5-60% DS granular starch.
 45. The process of claim38, wherein the granular starch is obtained from tubers, roots, sterns,fruits, seeds or whole grain.
 46. The process of claim 38, wherein thegranular starch is obtained from corn, cobs, wheat, barley, rye, milo,sago, cassava, manioc, tapioca, sorghum, rice or potatoes.
 47. Theprocess of claim 38, wherein the granular starch is obtained fromcereals.
 48. The process of claim 38, wherein the granular starch isobtained from dry milling or wet milling of whole grain.
 49. The processof claim 38, wherein the acid alpha-amylase and the glucoamylase arepresent in step (a) in a ratio of between 0.3 and 5.0 AFAU/AGU.
 50. Theprocess of claim 38, wherein the acid alpha-amylase and the glucoamylaseare present in step (b) in a ratio of between 0.3 and 5.0 AFAU/AGU. 51.The process of claim 38, wherein the acid alpha-amylase is an acidfungal alpha-amylase.
 52. The process of claim 51, wherein the acidfungal alpha-amylase is obtained from a strain of Aspergillus.
 53. Theprocess of claim 38, wherein the acid alpha-amylase is an acid bacterialalpha-amylase.
 54. The process of claim 53, wherein the acid bacterialalpha-amylase is derived from a strain of B. amyloliquefaciens, B.licheniformis, or B. stearothermophilus.
 55. The process of claim 38,wherein the glucoamylase is obtained from a strain of Aspergillus,Clostridium, or Talaromyces.
 56. The process of claim 38, wherein theglucoamylase is obtained from a strain of Aspergillus niger.
 57. Theprocess of claim 38, wherein step (a) is performed in the presence of aphytase.
 58. The process of claim 38, wherein step (b) is performed inthe presence of a phytase.
 59. The process of claim 38, wherein steps(a) and (b) are performed in the presence of a phytase.
 60. The processof claim 38, wherein step (a) is performed in the presence of acellulase and/or xylanase.
 61. The process of claim 38, wherein step (b)is performed in the presence of a cellulase and/or xylanase.
 62. Theprocess of claim 38, wherein the period in step (a) is between 20minutes and 1¼ hours.
 63. The process of claim 38, wherein the period instep (a) is between 20 minutes and 1 hour.
 64. The process of claim 38,wherein the period in step (a) is between 40 minutes and 1 hour.
 65. Theprocess of claim 38, wherein the period in step (a) is between 50minutes and 1 hour,
 66. The process of claim 38, wherein thefermentation in step (b) is for a period between 20 and 250 hours.